Disordered peptides and proteins are known to occupy the left-handed polyproline II (PII) conformation a significant fraction of the time. In spite of these insights, neither the residue-specific bias for PII, nor the role of PII in determining the stability of the native state is understood. The current project uses isothermal titration calorimetry (ITC) and NMR chemical shift perturbation to study a series of substituted peptides, which have been selected to provide access to the amino acid specific propensities to adopt the PII conformation. Specifically, the project takes advantage of the ability of SH3 domains to recognize and bind peptides in the PII conformation. As the PII conformation is helical, with three residues per turn, two of every three residues on the peptide are involved in binding to the SH3 domain, and the third residue is on the backside of the helix pointing into solution and making no contact with the SH3 domain. By studying substitutions at these non-interacting sites, the intrinsic bias of each amino acid can be illuminated. The studies conducted in this project will provide; 1) a calorimetrically based PII propensity scale, 2) a thermodynamic dissection of the component enthalpy and entropy contributions to the bias for each amino acid, and 3) the effects of common perturbants on the propensity. As such, this project provides a comprehensive thermodynamic description of the denatured states of proteins and peptides.
A quantitative characterization of the structure and energy of the denatured states of proteins represents the cornerstone to a molecular-level understanding of both protein stability and fold-specificity. Recent studies have revealed a significant bias in unstructured peptides towards the polyproline II (PII) conformation, even at non-proline residues. This indicates that the PII conformation is a dominant component of the denatured states of proteins. This project provides a comprehensive thermodynamic description of the denatured state, which represents a new potential source of data and information that will be especially useful in understanding the mechanism of protein folding. This project will provide training to students in thermodynamics, protein structure and folding, and a variety of biophysical techniques.
